Regulation of gonadotrope signaling and gonadotropin gene expression are central to the pathophysiology and treatment of a wide spectrum of diseases, including infertility, polycystic ovarian syndrome and gonadal hormone-dependent tumors. The major regulator of the gonadotropin genes LH and FSH is GnRH, which is released in short pulses the frequency of which varies during the female reproductive cycle. LH and FSH are differentially induced by GnRH, depending on the pulse frequency. Based on our recent discovery of several new secreted peptides that differentially regulate gonadotropin genes and on results of mathematical modeling, we hypothesize that control of gonadotropin gene expression and its frequency decoding is an emergent property of a distributed regulatory network including both intracellular signaling pathways and extracellular autocrine/paracrine factors. We plan to pursue a comprehensive study of the gonadotrope secretopeptidome and its regulation to identify new gonadotropin gene regulatory mechanisms and to develop a mathematical model to provide a comprehensive, testable and predictive framework for integrating the research in this field. We propose the following Specific Aims: 1. Characterize the gonadotrope secretopeptidome and study its regulation. iTRAQ secretopeptidome measurements from gonadotrope cell lines obtained under conditions that differentially regulate LH and FSH expression will be correlated with transcriptome assays using RNA-seq. Gonadotropin gene regulators will be selected by statistics and by a new computational approach we propose to develop that will infer subnetwork regulation from simultaneous analysis of these new gene expression and peptidomic datasets in the context of Bayesian integration of public datasets. 2. Study key secreted peptides and their mechanisms in gonadotrope regulation. Our initial secretopeptidome and RNAi experiments have identified several novel autocrine gonadotropin regulatory factors. The role of each factor in frequency- dependent regulation of gonadotropin secretion will be explored. Additional secreted gonadotropin gene regulatory candidates identified in Aim I will also be studied. 3. Develop and experimentally validate a model for gonadotropin gene frequency control. Several mechanisms for gonadotropin frequency decoding have been proposed and new regulatory loops are emerging from our work and that of other researchers. These mechanisms will be evaluated through a cycle of formal mathematical modeling, prediction, biological experiment and model refinement. Overall, this research program has the potential to identify and to characterize the mechanisms of action of new extracellular regulators of gonadotropin gene expression and to establish a model for GnRH frequency decoding and gonadotropin gene regulation.